Abstract
Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced at the infection site in response to pathogen invasion and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). The detection and quantification of camalexin content is required to understand how it is produced upon various stress conditions. Here we describe an easy method for camalexin extraction from Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea, and further determination of camalexin levels by liquid chromatography–mass spectrometry (LC-MS). The method is sensitive enough to trace amount of camalexin down to the low pico-gram (10 pg/mg FW) range.
Figure 1. The structural formula of camalexin
Materials and Reagents
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Representative data
Figure 2. Sample concentration. Evaporation is increased by passing an inert gas (N2) over the surface of the sample to remove the solvent. The gas travels through the gas chamber to the samples via the needles. A B Figure 3. LC-MS analyisis of camalexin from external standard (upper panels) and from the plant tissue (lower panels). A. Total ion chromatograms (TICs). B. ESI mass spectra in positive ionization mode. The mass of 201.048 corresponds to C11H9N2S, the protonated form of camalexin [M+H]+ (red arrows). Figure 4. Camalexin standard curve. A linear standard curve was obtained in the concentration range between 3.12 - 800 ng/ml.
Recipes
Acknowledgments
This work was supported by the European Research Council (advanced grant no. 233083). Part of the procedures were adapted from a previously described protocol for quantitative analysis of major plant hormones from crude plant extracts (Pan et al., 2010).
References
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